This Project will continue to study mechanisms that lead to tolerance of mature CD8+ T cells in the periphery. During the previous granting period we have defined two ways in which recognition of Ag can lead to tolerance, and we will continue to study these under the two Specific Aims. (Aim 1) To further elucidate the basis for the induction and reversibility of 'activation-induced non-responsiveness' (AINR). We have shown that CD8 T cells become anergic following a response to Ag and co-stimulation, and have termed this AINR to distinguish it from the classical 'anergy' that results from Ag recognition without helper- dependent regulatory checkpoint in the CTL response. In the absence of help AINR renders the cells tolerant. Several hypothesis regarding the function and fate of INR cells will be tested. (Aim 2) To determine the basis of peripheral tolerance induced by exposure to Ag in the absence of 'signal 3', and further characterize the tolerant cells with respect to phenotype, signaling defects, biological function and reversibility of tolerance. We have demonstrated that naive cells require 3 signals for full activation; TCR engagement (signal 1), co-stimulation (signal 2), and IL-12 (signal 3). In vivo exposure to Ag in the absence of 'signal 3' results in weak clonal expansion and no effector function, and the cells are rendered profoundly tolerant. Experiments are planned to test the biological relevance of this form of tolerance in models of tumor, autoimmunity and transplantation, and to elucidate the mechanistic basis of the tolerance. The planned experiments will include both in vitro studies using novel technology that we have developed for constructing artificial APCs and in vivo studies using the powerful approach of adoptive transfer of TCR transgenic T cells pioneered by Jenkins. We anticipate that the results of these studies will result in a better understanding of the fundamental mechanisms that govern tolerance induction in CD8 T cells, and will suggest novel approaches for immunization and immunotherapy to avoid or reverse tolerance.